Triclosan (TCS) is an antimicrobial that is used widely in hospitals, consumer goods, and personal care products such as hand soaps at concentrations ~10 mM leading to wide exposure at high concentrations to consumers. Mast cells are critical players in numerous diseases, including allergy, asthma, autoimmunity, infectious disease, cancer, inflammatory bowel disease, and even many central nervous system disorders such as autism, anxiety, and multiple sclerosis. Due to the centrality of mast cells in myriad physiological processes and diseases and the ubiquitous exposure of the U.S. population to this chemical, there is an urgent need for information on the mammalian toxicology and pharmacology of TCS. The Gosse lab has shown that TCS inhibits both degranulation (in rat RBL-2H3 cells and human HMC-1 cells) and actin cytoskeletal rearrangement. Both of these processes are stimulated by both antigen via the Immunoglobulin E (IgE) receptor Fc?RI, and by Ca2+ ionophore. This project will investigate the cellular and molecular mechanisms by which TCS suppresses mast cell degranulation and F-actin ruffling via Protein Kinase C (PKC) and the actin cytoskeleton and also through Phospholipase D (PLD) and microtubule transport. The hypothesis is that TCS affects the activity and cellular localization of PKC and actin cytoskeleton. PKC activity will be measured through an ELISA. Initial confocal microscopy experiments will define TCS effects on general sub-cellular localization and dynamics of PKC, actin, and related proteins in live mast cells. Next, super-resolution imaging with fluorescence photoactivation localization microscopy (FPALM) will be used to investigate PKC:actin and other protein binding interactions not visible in traditional microscopy. Another hypothesis being studied is that TCS affects the activity and cellular localization of PLD and microtubule transport Using both confocal and FPALM, TCS effects on tubulin dynamics and PLD localization will be investigated. A new method that utilizes fluorescent dextran as a reporter will be used to image TCS effects on the final step of degranulation to directly quantify the locations and timeframes of degranulation. This research will fill in missing knowledge on the effects of TCS on mammalian signaling, and will allow prediction of TCS effects in disparate cell types that share common signal transduction elements. The results of this study will also fulfill an urgent need by providig insights into the impact of TCS on human health.